Dynamics of changes in apical cell area during sex comb rotation in Drosophila melanogaster.

Epithelia are highly dynamic tissues displaying various types of tissue rearrangements (Weliky and Oster, 1990; Taylor and Adler, 2008; Harris and Tepass, 2010; Lee et al. , 2013; Firmino et al. , 2016; Rupprecht et al. , 2017). Here, we describe the dynamics of changes in apical cell area (ACA) in an epithelial system displaying tissue rearrangement resulting in sex comb rotation on the forelegs of male Drosophila melanogaster . The sex comb is a row of leg bristles which rotates during morphogenesis (Atallah, 2008; Atallah et al. , 2009; Malagon, 2013). We quantified the ACA in the region proximal to the developing sex comb by tracing apical cell boundaries using ImageJ in pupal first leg imaginal discs. We found that cells display intricate irregular oscillations in size as the comb rotates. However, the net changes in ACA within most of the cells studied are subtle, only 0 to +/-15%. Our current working hypothesis suggests these irregular oscillations confer flexibility during tissue rearrangement and can be an important mechanism for tissue homeostasis.

. Changes in apical cell area (ACA) during sex comb rotation in Drosophila melanogaster 12/13/2022 -Open Access

Description
Epithelia are sheets of interconnected cells, which are fundamental tissue structures in animal morphogenesis (Harris and Tepass, 2010). In recent years, the study of epithelial morphogenesis has incorporated the use of live imaging (Weliky and Oster, 1990;Taylor and Adler, 2008;Lee et al., 2013;Firmino et al., 2016;Rupprecht et al., 2017), showing that epithelia are highly dynamic tissues, requiring the interaction of multiple cellular processes including exchanging of cell neighbors (Taylor and Adler, 2008), changing in cell number, size and shape (Marinari et al., 2012;Moreno et al., 2019). However, despite the rapid progress in this field, it is still unknown how epithelia coordinate and buffer tissue rearrangement, while maintaining its functions as a physical barrier. An excellent model system for studying the dynamics of tissue rearrangement is sex comb rotation in Drosophila melanogaster (Malagon, 2013). The sex comb is a linear row of modified bristles located on the forelegs of D. melanogaster males (Atallah et al., 2009;Kopp, 2011). During pupation, the sex comb rotates from a perpendicular to a parallel position relative to the leg's long axis (Atallah et al., 2009;Kopp, 2011) (Fig. 1A-B). Previous work has shown that as the comb rotates, epithelial cells proximal to the sex comb rearrange displaying cell extrusion, exchange of cell neighbors, and changes in apical cell area. Here, we report that intricate irregular oscillations in cell size take place during tissue rearrangement. This variation in cell size during development can confer flexibility for tissue rearrangement without affecting epithelial integrity as a continuous barrier.
To investigate the dynamics of changes in apical cell area, we imaged wild type D. melanogaster legs using the line UbiDEcadherin::GFP which outlines cell boundaries (Oda and Tsukita, 2000). Then we manually outlined each cell through time using the imaging software, ImageJ. Later, we measured the changes in apical cell area (ACA) in the cells proximal to the sex comb (Malagon, 2013). Previous work showed that from a tissue perspective, the proximal region reduces to 1/2 of its original apical from cell rearrangement and elimination which results in comb rotation (Malagon, 2013) (Fig.1D). In addition, similar to other epithelial systems displaying tissue rearrangement via cell elimination, the epithelium proximal to the sex comb becomes more homogeneous in cell size 15 (Fig. 1D).
We found that although convergence in cell size occurs as previously reported (Malagon, 2013) (Fig. 1D-E), the cell dynamics to achieve tissue homogeneity display more complex temporal patterns than expected (Fig. 1F). In the three movies analyzed, cells display intricate irregular oscillations in ACA (Fig. 1F and Mov. 2 and 3 are shown in Fig. S1) To dissect this intricate pattern in more detail, we constructed the following two types of fate maps: 1) net changes in ACA (Fig. 1H-J) and 2) variation in ACA of each cell during rotation (Fig. 1K-L). In both types of fate maps, there are some commonalities as well as differences among movies. In particular, most of the differences are found in cells close to cells of individual bristles. The highest values of net reduction in ACA and standard variation are found in the cells located farthest from the sex comb (Fig. 1H-J). In addition, close to the sex comb, changes in ACA are generally very subtle (Fig. 1H-J). However, there is not a linear correlation between net changes in cell size and its variation during development (R 2 Mov. 1=0.37,Mov. 2 =0.30,Mov. 3=0.27). The smallest cells tend to have intermediate values of standard variation (Fig. S3).
Altogether, these findings in the dynamics ACA are consistent with previous studies in tissue rearrangement. For example, the complex patterns observed in the changes in ACA were also observed in other cellular processes such as exchange of cell neighbors (Levayer, Hauert and Moreno, 2015) and cell death (Marinari et al., 2012;Malagon, 2013). Our current working hypothesis is that many cells display a small net change in ACA; however, a high variation in cell size can offer flexibility to 12/13/2022 -Open Access the tissue to selectively eliminate cells without opening a gap in the epithelia. Future studies will need to test whether intricate patterns in apical size also take place in other systems, and its implications for tissue homeostasis and cancer formation.

Methods
Three time-lapse videos of wild type male D. melanogaster were recorded using confocal microscopy and analyzed in this study following the protocol described by Atallah (2008) 15 . For live imaging, pupae were mounted in halocarbon oil (series 700; Halocarbon Products) on a coverslip (Sigma) and imaged with a laser 510 scanning confocal microscope (ZEISS model) at 25 degrees with a 40× objective, using LSM Browser software (ZEISS). Z-stacks had a 3 μm step size.
Analysis of these three videos was performed using the ImageJ software (NIH, http://rsb.info.nih.gov/ij). 2D projections were generated, then epithelial cells proximal to the sex comb were manually outlined and followed through the time of 23 to 36 hours after pupation (AP) (Fig. S3). We labelled more than 40 cells per movie (Mov. 1=44 cells,Mov. 2 = 51,Mov. 3 =55), depending on how easy it was to accurately label cells during morphogenesis. If cells were not easy to distinguish, we did not collect data for that specific time point. Cells that were eliminated during tissue rearrangement were not included in the analysis. The changes in the apical cell area were measured using the subroutine "Analyze Particles" function of the ImageJ software (NIH, http://rsb.info.nih.gov/ij). Error bars represent +/-1 standard deviation.

Fate maps
The net changes in ACA were calculated by subtracting the final time point (36 hours AP) from the initial time point (23 hours AP). The variations in ACA were calculated using the standard deviations.

Reagents Fly strains
The ubi-DEcad::GFP lines were generated by Oda and Tsukita (2000), and I obtained copies from the Ulrich Tepass laboratory. Flies were reared on a yeast-cornmeal-molasses medium at 25°C.